Field of the Invention
The present invention relates generally to paracrine signal measuring and, more specifically, to label-free mapping of single cell secretions in real time.
Description of the Prior Art
Paracrine signaling is a form of close-range communication between cells, typically mediated by the secretion of proteins. The types of proteins secreted as well as their spatial and temporal distributions give rise to a broad range of possible responses amongst the receiving cells, including cell migration and proliferation. Not surprisingly then, paracrine signaling is found to play a central role in a diverse range of processes such as wound healing, angiogenesis and immune response, which rely heavily on cell movement and division. The ability to map the spatio-temporal nature of individual cell secretions is thus foundational to understanding these processes.
There are, however, a number of roadblocks encountered in trying to measure paracrine signaling due to the proteins being both highly localized and external to the cell. While fluorescent fusion protein tags are now standard for tracking intracellular signaling, the approach is problematic for studying secreted proteins. First, the presence of a relatively large tag (27 kDa for GFP) may hamper the cell's ability to secrete the protein of interest. Second, even if the molecule and its fluorescent protein tag are successfully secreted, the result is a diffuse glow in the vicinity of the cell that is difficult to track quantitatively in space and time.
As a result, direct measurements of secreted proteins from individual cells are typically performed using techniques founded upon immunosandwich assays that either use fluorescent antibodies or colormetric enzymatic reactions. While in the past such measurements had time resolutions on the order of days, technological advances that couple immunosandwich assays with lithographically patterned microwells and microfluidics have enabled quantitative secretion monitoring with time resolutions on the order of hours. Such advances have exposed cyclical behaviors in the rates at which stimulated T cells secrete cytokines, and in a more general sense, have demonstrated how improving time resolutions can enhance the understanding of intercellular signaling. Improved temporal resolutions hold the promise of detecting the time for individual cells to begin secretion after external stimulation, correlating secretion rates with stages of the cell cycle and, as we show in the present invention, distinguishing burst-like secretions from those that are more steady state in nature. Immunosandwich-based assays are now capable of measuring hundreds or thousands of individual cells per experiment but their temporal resolution is limited by the introduction of the antibody probe which necessarily halts or ends the secretion study. A complimentary technique that focuses on a small number of cells but with higher spatial and temporal resolution promises to help complete the picture of close range cell-to-cell communication by bridging the time scale gap from seconds to days.